HPLC is established as an efficient tool in both analytical and preparative techniques for the recovery of biological products in high purity. Reversed HPLC with bonded supports (such as C.sub.18 -silica) is optimal for highly polar solutes. Reversed phase HPLC generally involves the use of a polar eluent with a non-polar support. Highly polar solutes have a greater affinity for the eluent than does the support and solutes are eluted in reverse order of polarity.
High molecular weight biological molecules such as proteins have a complex three dimensional structure which must be preserved to maintain the biological function of the sample. In a reversed phase chromatographic separation, the optimal separation involves the interaction between the hydrocabonateous groups of the support and hydrophobic surface features on column is therefore related to specific topological features of the individual protein species, with the components of lower or less accessible hydrophobic surface patches being eluted first. Because of this separation mechanism, HPLC is a highly resolutive chromatographic method. Even slight compositional differences between proteins will lead to topological differences which can result in an effective separation. For example, porcine and human insulin are easily separated by HPLC, although the two molecules only have a minor difference in a single amino acid residue.
Hydrophobic interactions between a target protein molecule and the reversed phase column must be carefully controlled. If the protein molecule is significantly destabilized in the chromatographic medium, unfolding of the three dimensional structure of the protein molecule can expose buried hydrophobic groups which can lead to additional hydrophobic interactions with the reversed phase column and prevent elution of the protein. Such increased interactions between the solute and stationary phase can lead to denaturation of the protein molecule and/or low recoveries due to irreversible multisite binding of the solute.
In addition, there are different requirements in conducting HPLC on a preparative scale from those in conducting HPLC on an analytical scale. The protein must not itself be denatured in a preparative process and it must not pick up any contaminants from either the mobile phase or the support at any time while it is passing through the column. Mobile phases used in analytical scale processes often destabilize the protein sample and thus are not suitable for preparative separations. In addition, solvents such as acetonitrile are widely used to elute polypeptide samples that are retained by reversed phase columns. Such solvents are highly toxic and cannot be used in preparative separations.
In reversed phase chromatography of proteins the solvents acetonitrile, isopropanol or propanol are used widely. Mixtures of phosphoric acid and amines and perfluorinated carboxylic acids, especially trifluoroacetic acid (TFA), are popular as ionic modifiers to be added to the mobile phase. While in analytical HPLC these solvents and buffer systems are acceptable, in preparative chromatography the solvents and ionic modifiers must be non-toxic and easily removed and some of the analytical reagents are not suitable for this reason.
The mobile phase for use in preparative HPLC should be mild to the protein so as not to denature it and be inert to the support. Damage to the support is not only inherently disadvantageous in relation to the support itself but can produce toxic by-products which are not acceptable with proteins which might be used for, for example, pharmaceutical applications.